Our goal for the combined R21 and R33 phases of this proposal is to make an integrated fluidic device that is capable of the automatic isolation of target cells from a cell mixture and their analysis for molecular markers for cancer detection and diagnosis. The final integrated device will be comprised of the following stages: (1) a cell separator able to process 2x106 cells/minute and to trap suspect tumor cells from them; (2) a cell fractionator that will separate suspect cells according to their dielectric and surface marker properties; (3) a microfluidic isolator stage that will collect and concentrate cell subpopulations emerging from the fractionator, combine them with a mixture of beads carrying multiple molecular probes, and burst them in the presence of the beads; and (4) an impedance sensor stage able to identify beads according to their dielectric fingerprints and thereby index them to the probes carried on their surfaces. Fluorescence of molecular probes on each bead will also be measured at this stage. By using a mixture of multiple bead types, several molecular assays will be run simultaneously. To achieve this goal we will develop and integrate fluidic and microfluidic technologies to automate the central problems of molecular diagnosis, namely the enrichment and isolation of a suspect cell subpopulation from a mixture of cells in a biopsy sample and the quantitative analysis of that subpopulation for molecular markers including proteins and mRNA's. The key technologies to be developed are: 1) A prefilter able to trap suspect tumor cells from large numbers of blood or lymph node cells; 2) A force balance method that exploits the dielectric and density properties, and, optionally, the immunomagnetic labeling properties, of target cells to enable the isolation; and 3) A dielectric indexing and manipulation method for carrier beads that, when combined with established molecular assays, will allow the simultaneous quantification of multiple molecular markers in parallel assays. The sample preparation aspects represent an extension of our successful dielectrophoretic trapping and dielectrophoretic field-flow-fractionation (DEP-FFF) methods that have been demonstrated for sorting and isolating different cell types from a cell mixture. The dielectric bead methods represent a novel approach to identifying individual molecular tests in a parallel molecular marker analysis scheme. Dielectric indexing of carrier beads will be used instead of the spatial indexing used on a gene chip. This will allow different subpopulations of beads, each carrying a different marker probe, to be identified and differentially manipulated by exploiting their dielectric signatures. The need to immobilize molecular probes in a tightly specified location on a fixed substrate, as demanded by spatial indexing is thereby eliminated and customized mixtures of probes, each carried on a separately-indexed bead type, may be identified as they emerge in a mixture from a reusable assay system. The study will be divided into a one year R21 phase and a three year R33 phase. During the R21 phase, proofs-of-concept will be developed for scaled-up dielectrophoretic trapping of suspect cells from large cell populations, for combined dielectrophoretic-magnetophoretic field flow fractionation, and for dielectric indexing of beads in mixed molecular assays. During the R33 phase, these technologies will be further developed and integrated in order to accomplish all steps of sample preparation and molecular analysis. The technologies will then be tested and refined using clinically relevant samples.